Chloride channels join the sperm ‘channelome’

A human sperm is a simple cell with a complex mission. From the vagina it must progress through the cervix and uterus to the oviduct, then ascend to the site of fertilisation, where it must find, recognise and interact successfully with the oocyte. This requires that the cell performs complex regulation of its motility, undergoes capacitation (a process of maturation that confers fertilising capacity) and acrosome reaction (exocytosis of the acrosomal vesicle) and binds to and penetrates the egg's vestments. Sperm neither transcribe new mRNA nor translate existing mRNAs so post-translational modification of ‘inherited’ proteins is crucial. Kinases and phosphatases are central players in this regulation but ion channels and ionic gradients are pivotal, orchestrating the maturation and activities of the cell, controlling and integrating both rapid and more prolonged changes (Darszon et al. 1999).

Until recently application of electrophysiological techniques to sperm, allowing direct measurement of the activity of these channels, has been prohibitively difficult. Getting ‘good’ seals between patch electrodes and sperm plasmalemma had an unacceptably low success rate and obtaining whole cell access proved impossible, the primary problem being the minimal cytoplasmic volume and consequent ‘tight’ fit of the sperm's membrane over the nucleus and flagellum. In 2006 the problem was finally overcome and the first report of successful whole-cell patching of mouse sperm was published. Testicular and epididymal mouse sperm have some retained cytoplasm (cytoplasmic droplet) on the flagellum, which is no longer present after ejaculation. By placing their pipette on the small cytoplasmic droplet of epidiymal sperm, Kirichok et al. (2006) successfully sealed and obtained whole-cell access (confirmed by filling of the cell with fluorescent dye). In human cells the cytoplasmic droplet is situated at the neck region of the cell and is retained after ejaculation. Lishko et al. (2010) successfully patched this droplet and described whole cell records from mature, ejaculated human sperm. The first sperm ion channel current to be characterised was CatSper, a sperm-specific, Ca²⁺-permeable cation channel. Catsper has a tetrameric VOCC-like structure, but is highly sensitive to elevated pH_i, which is a key regulatory factor in sperm. Subsequently a pH_i-regulated K⁺ channel (KSper, Slo3), a voltage-sensitive proton channel (Hv1; believed to play a key role in regulating pH_i of human sperm) and a ligand (ATP)-gated current (P2X2) of mouse epididymal sperm have been described. By triturating the cells to separate heads and tails it has been possible to show that all four of these conductances are localised to the flagellar principal piece or midpiece (Lishko et al. 2012).

In this issue of The Journal of Physiology, Orta et al. (2012) have provided two important contributions to this rapidly moving field. Firstly, this is the first characterisation of functional anion channels in sperm. They present convincing evidence for expression of a calcium-dependent chloride channel (CaCC) and show that TMEM16A is a likely constituent of the channel. Roles for chloride fluxes in regulation of E_m, volume, pH and [HCO₃⁻]_i (crucial for controlling sperm's soluble adenyl cyclase) are well established and extracellular chloride is required for sperm to capacitate. Orta et al. also show that the ability of sperm to undergo the acrosome reaction (a prerequisite for fusion with the oocyte) shows similar pharmacological sensitivity to the CaCC described here, emphasising the importance of Cl⁻ fluxes in this process.

Secondly, they describe a new technique for perforated patch recording from the sperm head. Initial outcomes from whole cell patch clamp of sperm have been both fascinating and surprising. Data from immunochemical and pharmacological studies suggested that many ion channel types from somatic cells are present in sperm and involved in regulating function, yet only a handful of conductances have been detected so far in mature and epdidymal sperm (Lishko et al. 2012). It is likely that only a fraction of the channels reportedly present in sperm are functional – some may be switched off/removed during maturation (Darszon et al. 2011) and some may have been misidentified – for instance CatSper responds to various stimuli (voltage, pH_i, small organic molecules including odorants and cell-permeant cAMP analogues) and its activity may have been interpreted as evidence of other cation-permeable channel types (Brenker et al. 2012). However, though perforated patch records of CatSper current have been obtained from mouse sperm cytoplasmic droplet (Kirichok et al. 2006), it should be remembered that most work has been done by standard whole cell patching. Many proteins in sperm are strongly regulated by balanced activities of kinases and phosphatases so a procedure that appears rapidly to dialyse this minute cell may have profound effects on cell physiology, including silencing of a number of channels. We know from cell-attached patching on the head of intact sperm (Darszon et al. 1999; Jimenez-Gonzalez et al. 2007) that several types of channels are present here. These cannot be CatSper, Slo3, Hv1 or P2X2, which are flagellar. The technique described by Orta et al. allows whole cell recording from the head and (hopefully) minimises any effects of dialysis or structural effects that may occur during patching the cytoplasmic droplet. Significantly, the CACC described by Orta et al. appears similar whether recorded through the head or the droplet, but we must wait to see if perforated patching of the head and consequent preservation of the cytoplasm will reveal the presence of more ‘sensitive’ channels.